The work described here is designed to study the mechanisms involved in regulation of pre-mRNA splicing. One aspect of this regulation is alternative splicing of 5' and 3' splice sites on pre-mRNAs to generate multiple isoforms of proteins with distinct functions from single genes. Although this level of gene expression is ubiquitous in eukaryotes the mechanisms that regulate alternative pre-mRNA splicing are not well understood. We have found that a family of pre-mRNA splicing factors, called SR proteins, are involved in the regulation of alternative pre- mRNA splicing. Biochemical and genetic experiments proposed in this application are designed to better understand how SR proteins regulate pre-mRNA splicing. One question we hope to answer is, when during spliceosome assembly do SR proteins exert their function to specify splice sites. We hope to answer this by observing the sequential, splicing-dependent recruitment of U-snRNPs to two different 5' splice sites using SR proteins with different site preferences. We also hope to pursue the regulation of SR protein function by phosphorylation because we have found that only the phosphorylated form of SR proteins can initiate pre-mRNA splicing. Our previous results have led us to hypothesize that independent regulation of SR protein expression in a given nucleus plays a role in specifying alternative splicing. We propose to test this by examining splice site selection in cells where we have altered the levels of particular SR proteins. Finally, we propose to address the distinct functions of each SR protein in vivo through a genetic analysis of SR genes in the nematode C. elegans. This organism is well-suited to genetic analysis because of reverse genetic approach has recently been developed that allows for targeted inactivation of genes.